Towards next-generation diagnostic pathology: AI-empowered label-free multiphoton microscopy

Diagnostic pathology, historically dependent on visual scrutiny by experts, is essential for disease detection. Advances in digital pathology and developments in computer vision technology have led to the application of artificial intelligence (AI) in this field. Despite these advancements, the variability in pathologists’ subjective interpretations of diagnostic criteria can lead to inconsistent outcomes. To meet the need for precision in cancer therapies, there is an increasing demand for accurate pathological diagnoses. Consequently, traditional diagnostic pathology is evolving towards “next-generation diagnostic pathology”, prioritizing on the development of a multi-dimensional, intelligent diagnostic approach. Using nonlinear optical effects arising from the interaction of light with biological tissues, multiphoton microscopy (MPM) enables high-resolution label-free imaging of multiple intrinsic components across various human pathological tissues. AI-empowered MPM further improves the accuracy and efficiency of diagnosis, holding promise for providing auxiliary pathology diagnostic methods based on multiphoton diagnostic criteria. In this review, we systematically outline the applications of MPM in pathological diagnosis across various human diseases, and summarize common multiphoton diagnostic features. Moreover, we examine the significant role of AI in enhancing multiphoton pathological diagnosis, including aspects such as image preprocessing, refined differential diagnosis, and the prognostication of outcomes. We also discuss the challenges and perspectives faced by the integration of MPM and AI, encompassing equipment, datasets, analytical models, and integration into the existing clinical pathways. Finally, the review explores the synergy between AI and label-free MPM to forge novel diagnostic frameworks, aiming to accelerate the adoption and implementation of intelligent multiphoton pathology systems in clinical settings.


Introduction
Pathology often provides the "gold standard" for disease diagnosis 1 .Historically, this discipline has relied on the keen eyes of pathologists to make clinical judgments based on visual examinations of stained tissue sections under a microscope to classify diseases and determine their prognoses.The advent of whole slide imaging (WSI) scanners in the last decade has transformed how these images are collected and examined, ushering in a new era for pathology 2 .WSIs have become a cornerstone for remote pathology consultations, routinely facilitating diagnosis, research, and education in pathology, offering unprecedented convenience to practitioners.However, the reliance on the extensive expertise of experienced pathologists persists, whether reviewing slides under a microscope or analyzing WSIs.The training cycle for professional pathologists remains lengthy and demanding.This factor, coupled with the growing number of cases needing diagnosis annually, poses an escalating challenge for medical institutions striving to maintain high-quality diagnostic services.
Artificial intelligence (AI), predominantly driven by deep learning, has shown its superiority in various computer vision applications including image enhancement [3][4][5] , classification 6 , detection 7 , and segmentation by automatically recognizing and extracting complex features from images 8 .Simultaneously, the availability of large-scale WSI datasets rich in pixel-level detail has allowed the expansion of deep learning techniques, traditionally applied to natural images, to the realm of microscopic imagery.The digital transformation of clinical pathology has led to the automation of various aspects of the field, integrating supportive diagnostic techniques such as diagnosis 9 , biomarker identification 10 , and prediction 11 , which combinedly to be computational pathology.
The WSI-based intelligent pathology not only alleviates the burden on pathologists but also provides both patients and clinicians with more objective tools for diagnosis and prognosis.Nevertheless, diagnostic procedures within pathology still face a "gray zone", where varying interpretations of diagnostic criteria among pathologists can lead to discrepancies in the diagnosis of certain conditions.The precision required for personalized cancer therapy further escalates the need for accurate tissue pathology marker diagnosis, whereas misdiagnoses can lead to misguided treatments and can also hinder the progress of drug development.Consequently, to develop a new generation of pathological diagnostic paradigms, it is essential not only to create an AI-assisted diagnostic framework but also to incorporate innovative multimodal imaging techniques that enhance conventional pathology.
The most commonly pathological staining method is hematoxylin and eosin (H&E) staining.Irrespective of intraoperative frozen sections or postoperative paraffin sections, the production of H&E slides involves intricate histological procedures such as biopsy, fixation, sectioning, and staining.With the advancement of label-free optical microscopy 12,13 , techniques such as quantitative phase imaging (QPI) 14,15 , photoacoustic microscopy (PAM), optical coherence tomography (OCT) 16 , and stimulated Raman scattering (SRS) microscopy have complemented traditional pathology.They offer unique insights into cellular physical parameters in vitro, functional imaging in vivo, and tissue molecular characteristics.Notably, multiphoton microscopy (MPM) enables simultaneous imaging of multiple intrinsic components within biological tissues.Moreover, it attains imaging contrast and resolution comparable to traditional histopathology, directly extracting qualitative microstructure and quantitative spectral features for pathological diagnosis 17 .Enabled by deep learning methodologies, OCT facilitated the automated detection of geographic atrophy in age-related macular degeneration 18 .QPI allowed for virtual quantitative fluorescent imaging of live organoids 19 , while PAM allowed intraoperative histology of bone tissue 20 .Additionally, SRS provided near real-time intraoperative diagnosis of brain tumors, creating a complementary diagnostic pathway independent of traditional pathology laboratories 21 .These technologies significantly enhance the accuracy and efficiency of diagnosis.For AI-empowered MPM, comprehensive exploration of multiphoton feature patterns, such as tumor infiltration patterns [22][23][24][25] and vascular collagen deposition 17,26,27 , has been integrated to achieve distinctive auxiliary diagnosis [28][29][30][31][32][33][34][35][36][37][38][39][40][41] and prognosis prediction 42 capabilities, holding great promise for clinical translation.
In this review, we first provide a concise overview of multiphoton physics mechanisms and multiphoton microscopic instrument.Subsequently, we systematically summarize pathological applications of MPM in various human diseases.Drawing on multiphoton pathological imaging, we explore the positive impact of artificial intelligenceextending from machine learning to deep learningin advancing diagnostics assisted by multiphoton pathology.Finally, considering the current status of multiphoton intelligent pathology and the requirements for precision diagnostics, we discuss the challenges and future perspectives associated with the integration of MPM and AI.We anticipate that this review will contribute to the clinical translation and intelligent applications of multiphoton microscopy, fostering progress in "next-generation diagnostic pathology".

Label-free multiphoton microscopy
Label-free optical microscopy exploits the interaction of light with biological tissues, such as refractive index, molecular vibrations, scattering, or absorption, to achieve various imaging contrasts.Table 1 provides a comparative overview of the capabilities and applications of common label-free biomedical microscopy.The diverse imaging mechanisms of these techniques render them suitable for different clinical applications.QPI measures phase changes to obtain contour and morphology information of in vitro cell samples.This computational-based optical system is both simple and cost-effective.PAM and OCT achieve imaging depths at the millimeter scale.Although they sacrifice some spatial resolution, there in vivo vasculature and ophthalmology applications are also clinically recognized.
To obtain high-resolution, high-contrast images resembling those produced by traditional pathology, two nonlinear optical microscopies, MPM and SRS have been widely applied in label-free pathological diagnosis.SRS not only acquires pathological images but also enables selective Raman spectral analysis of components such as lipids and proteins.However, the complexity of the excitation light source module in current SRS systems has hindered its widespread commercial adoption, and further exploration is needed to fully establish its indications for pathological diagnosis.In contrast, commercial multiphoton microscopes, based on SHG and TPEF, have   reached a high level of maturity and availability since their inception in 1997.MPM has been applied to examine tumor pathology in as many as 16 human organs, such as brain tumors 17,[43][44][45][46][47] , breast cancer 23,24,[48][49][50][51][52][53] , and colorectal cancer 30,[54][55][56][57] .Consequently, MPM was highlighted as one of the significant advancements in label-free histopathology in the 2016 research highlights of Nature Methods 58 .
The principle of multiphoton microscopy MPM requires high peak power from ultra-short pulse lasers.To capture a multiphoton image of a single field of view, the excitation light scans the specimen point-bypoint and line-by-line via scanning system and objective.When multiple low-energy photons simultaneously reach the fluorophores or specific structures in specimen, they interact to produce multiphoton optical signals, including two-photon/three-photon excited fluorescence and second/third harmonic generation.These signals are typically collected in an epi-detection configuration by the objective and guided onto the photomultiplier tubes, which convert the optical information into electrical signals.By utilizing an XY translation stage to sequentially capture images from each position within the specimen, a large-scale stitched image can be constructed.

TPEF
TPEF is a third-order nonlinear absorption process.In this process, a fluorescent molecule or atom simultaneously absorbs two photons of the same frequency.During the absorption process, electrons in the ground state are first excited to an intermediate "virtual state" by one photon and then further excited to the final excited state by another photon.In other words, absorption of two photons of the same frequency excites electrons to a higher energy level.Following a certain relaxation time, electrons in the excited state spontaneously transition back to the ground state, emitting fluorescence with a frequency slightly lower than twice the incident light frequency.

SHG
SHG is a second-order nonlinear optical phenomenon, also known as "frequency doubling".It refers to the output photons having twice the frequency of the incident photons when two photons of the same frequency interact with a nonlinear medium.The output second harmonic wave is termed the second harmonic.In the process of second harmonic generation, an electron in the ground state absorbs two photons of the same frequency, is excited to a virtual state, and then emits a second harmonic photon before returning to the ground state.

Endogenous signal sources
In biological tissues, numerous biomolecules exhibit TPEF and SHG signals.For instance, TPEF can image endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) 59 .SHG occurs in non-centrosymmetric molecular structures like collagen 60 , microtubules 61 , and myosin 62 .Thus, SHG and TPEF endogenous signals provides a comprehensive characterization of tissue structure and multi-parameter functional metabolism.This approach overcomes the influence of labeled biological processes or toxicity, offering a crucial tool for studying pathological samples.Taking the example of multiphoton imaging of cerebral vascular malformations, Fig. 1a illustrates images from the SHG and two TPEF detection channels, along with a schematic representation of endogenous signal sources 17 .Detailed endogenous signal sources have been summarized in previous references 63,64 .

Multiphoton microscopic instrument
Figure 1b illustrates the representative history of AIempowered label-free MPM [65][66][67][68][69][70][71][72][73] .In 1931, Maria Goeppert-Mayer proposed the concept of TPEF 74 .Thirty years later, the invention of the laser facilitated the first experimental verification of TPEF 75 .In 1974, Robert Hellwarth introduced the SHG microscope, utilized for observing spatial structural changes in ZnSe crystals 76 .In 1990, the Webb group introduced the concept of twophoton excitation fluorescence microscopy, marking DNA in pig kidney cells and observing chromosome morphology in live cells 65 .In 1997, the Bio-Rad company produced the first commercial multiphoton laser scanning microscope.Currently, globally microscope companies are continually innovating desktop multiphoton laser scanning microscopy, greatly advancing the life sciences 77,78 .
The commercial research-grade multiphoton microscopes, designed to meet the needs of most researchers, typically can simultaneously image both labeled and unlabeled specimens.However, the large equipment footprint of such microscopes requires placement on laboratory optical platforms, and their high cost discourages some users.Therefore, researchers have been devoted to developing more portable and economical multiphoton microscopes.Although the miniaturized design and integration technology may sacrifice image resolution or field of view, this trade-off makes the instrument more portable, suitable for applications in onsite pathology diagnosis and other widespread diagnosis scenarios.In 2017, fast high-resolution miniature twophoton microscopy was successfully applied to brain imaging in freely behaving mice 67 .In 2018, the multimodal label-free nonlinear imaging system was implemented to intraoperatively characterize the tumor microenvironment 52 .Excitingly, in 2023, the space station-level two-photon microscope achieved the first  Fig. 1 The development history of AI-empowered label-free multiphoton microscopy.a Schematic diagram of label-free multiphoton imaging, representative SHG/TPEF images, and corresponding endogenous signal sources 17 .b Historical timeline of multiphoton microscopic instruments 52,[65][66][67][68] , multiphoton pathological applications 25,27,[69][70][71] , and AI-empowered multiphoton intelligent pathology 31,41,42,72,73 .TPEF twophoton excited fluorescence, 3PEF three-photon excited fluorescence, SHG second-harmonic generation, THG third-harmonic generation three-dimensional images of astronauts' skin.For future challenges in the development of multiphoton microscopic instrument from research-grade to pathologicalgrade, please refer to Section 6.3.
With the continuous development of multiphoton instruments, there has been a significant emergence of pathological applications in MPM.Meanwhile, the rise of artificial intelligence technology enables multiphoton intelligent pathology.Section 4 introduces the applications of multiphoton pathology, while Section 5 focuses on AI-empowered multiphoton pathology diagnosis.

Applications of multiphoton microscopy in pathological diagnosis
Label-free MPM, with its specific identification of cellular cytoplasm, extracellular matrix, and their interactions, has opened a novel perspective in pathological research.This section summarizes the applications of MPM in pathological diagnosis through the exploration of multiphoton diagnostic features.
Firstly, multiphoton imaging of the cytoplasm reveals rich cellular morphological information, such as cancer cells 79 , hyperplasia 43,55 , and necrosis 44 , which is crucial for determining the grading and prognosis of tumors.Additionally, through the analysis of specific features of cancer nests, different tumor growth patterns 80 can be distinguished, providing a basis for the formulation of clinical treatment plans.It is noteworthy that MPM can also quantitatively reflect cellular metabolic activity by measuring the ratio of NADH to FAD in the cytoplasm 81 .In addition to cancer cell identification, MPM can differentiate other cell types, such as myoepithelial cells 82 , lymphocytes 37 , and glandular cells 83 , based on differences in cytoplasmic morphology and signal intensity.Taking myoepithelial cells as an example, this provides crucial features for challenging diagnoses such as microinvasive breast cancer.
Secondly, MPM exhibits high sensitivity to the extracellular matrix, especially collagen fibers 84 and basement membrane 80 .By analyzing the morphology of collagen fibers, different vascular patterns in tumors can be distinguished 43 , aiding in the assessment of malignancy and progression of tumors.For instance, observations of glomeruloid vessels in glioblastomas 85 , hyaline degeneration and collagen aging in cerebral cavernous malformations 17 .Furthermore, the quantification of fibrosis 45 and proliferative reactions 86 can be facilitated by extracting features of collagen fibers, which provides crucial evidence for disease progression.More importantly, combining information from the cytoplasm and extracellular matrix, MPM can observe diverse spatial distribution patterns, such as tumor-associated collagen signatures (TACS) 23 and tumor-infiltrating lymphocytes (TILs) 24 , offering a unique perspective on the occurrence and development of infiltrating tumors such as gastric cancer, colorectal cancer, and breast cancer.

AI-empowered multiphoton pathological diagnosis
Prior to the application of AI in multiphoton images, conventional digital image processing algorithms, such as collagen fiber analysis 132 and saliency detection 133 , were already in existence for the quantitative assessment of tumor-specific multiphoton diagnostic features.In this section, as shown in Fig. 4, we specifically focus on the work related to machine learning and deep learning, providing a brief overview of their empowered capabilities in pathological diagnosis, including image preprocessing, disease diagnosis, and prognosis prediction.
The quality of multiphoton images serves as a prerequisite for ensuring the accuracy of disease diagnosis and prognosis prediction.Therefore, before using multiphoton images for disease diagnosis, researchers often employ image preprocessing techniques, such as image restoration 73,134,135 and image super-resolution models [136][137][138] , to enhance the image textural details and restore hidden pathological features.For instance, to address image quality caused by uneven sample or system instability, adaptive sampling driven by the uncertainty of predicted pixels can be employed to reduce noise 134 .For stitched multiphoton images, stripe self-correction networks based on proximity sampling scheme can effectively correct stripes or artifacts in the stitched positions 135 .Additionally, a self-alignment dualattention-guided super-resolution network can produce high-quality multiphoton images while mitigating the risk of photobleaching 138 .These preprocessed high-resolution, high-contrast multiphoton images can further enhance the accuracy of downstream diagnostic tasks, such as cell segmentation and counting, and improve the precision of prognostic tasks related to the extraction of collagen features.
On the other hand, as multiphoton imaging gradually enters the field of pathology, virtual image generation techniques serve as a complementary form of preprocessing that enhances the acceptance of multiphoton pathology images.Virtual image generation encompasses the transformation from label-free multiphoton images to virtual pathological staining images 17,72,139,140 , as well as the generation of virtual multiphoton images from H&Estained images 141 .For instance, virtual staining models based on generative adversarial networks (GANs) 17 or convolutional neural networks (CNNs) 72,[139][140][141] can transform multi-channel multiphoton images into H&E staining or specific staining images.Although virtual staining images may sometimes deviate in detail from real stained images, these pathology-styled images assist pathologists in interpreting multiphoton images more effectively.Moreover, CNN architectures with pixel-shuffle layers can generate virtual SHG images directly from H&E-stained images 141 , eliminating the need for additional staining agents or equipment.This provides a cost-effective method for quantitatively extracting collagen fiber directionality and alignment features.These preprocessing steps provide the foundation for subsequent pathological analysis, facilitating more accurate diagnosis and prognosis.
Currently, diagnostic challenges persist in contemporary pathology, with specific scenarios proving particularly difficult to interpret with precision.Notable examples include the differentiation between glioblastoma Fig. 2 Representative multiphoton pathological atlas of different diseases.Multiphoton microscopy can be used for a variety of diseases in different tissues of the human body.These include tumor diseases such as glioblastoma, liver cancer 37 , hilar cholangiocarcinoma 95 , breast cancer 23 , ovarian cancer peritoneal metastases 94 , prostate cancer 93 , colorectal cancer 54 , oncocytoma 90 , intramural metastasis in esophageal squamous cell carcinoma 91 , ductal adenocarcinoma in the pancreatic head 92 , gastric cancer 89 , nonpapillary urothelial carcinoma 88 , and squamous cell carcinoma of the lung 87 .Additionally, it can be utilized for non-tumor conditions like morphea 28 , myocardial fibrosis 97 , and atherosclerotic lesions 96 .Schematic diagram of human body structure and organs are drawn by Figdraw.Adapted with permission from ref. 91,94 © Optical Society of America and primary lymphoma 142 , and between ductal carcinoma in situ and microinvasive carcinoma of the breast 82 .These diagnostic challenges share similarities, often cannot be addressed through conventional pathological techniques.For instance, H&E staining struggles to differentiate or accurately quantify vascular-related elastic fibers and collagen fibers.Additionally, specialized cells such as myoepithelial and basal cells are prone to confusion with neighboring proliferative fibroblasts in the stroma.Excitingly, MPM offers a promising solution that aids in the identification of ambiguous cells, which helps mitigate the subjective differences among pathologists.More importantly, the integration of AI introduces a level of objectivity, supplying auxiliary information that enhances the subjective visual diagnosis performed by human experts.
Researchers commonly utilize machine learning methods and deep learning models to automatically extract distinct features of cellular cytoplasm and extracellular matrix from multiphoton images.For instance, segmentation models based on U-Net are employed to extract multiphoton features such as elastic fibers 28 and cells 29 , enabling rapid detection and quantification of pathological regions.Besides, a novel diagnostic method has been developed by fusing the H&E segmentation results of cell nuclei with multiphoton images, leading to more accurate diagnoses of microinvasion in ductal carcinoma in situ 82 .The method of combining feature extraction methods with machine learning classifiers, such as support vector machine (SVM) 33 or stochastic gradient descent (SGD) 30 classifier, has shown superior performance in classifying diseases, particularly on small datasets.In contrast, using deep learning classification networks such as ResNet 34 or VGG 35 allows for the automatic learning of complex pattern.Prognostic prediction is of paramount significance for understanding disease progression and guiding patient treatment.A robust prognostic prediction model is often associated with the accuracy of pathological diagnostic results and the discovery of pathological novel insights.For instance, based on the tumor-associated collagen signature patterns revealed by MPM in invasive breast cancer, the integration of graph neural networks has facilitated a deeper interpretation of the spatial distribution of these patterns in tumor development 42 .This approach also provides new clues for the precise classification and treatment of different breast cancer subtypes.With the gradual accumulation of multiphoton datasets, AI-empowered MPM augments the dimensions and efficiency of traditional pathology, elevating multiphotonassisted diagnosis to a more intelligent and precise level, thereby assisting clinicians in improving the diagnostic accuracy of intractable cases.Table 3 provides a detailed summary of the model types, inputs, and outputs involved in representative label-free multiphoton image preprocessing and intelligent pathological diagnosis from 2013 to 2023 17,[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]72,73,[134][135][136][137][138][139][140][141] . Fig. 3 Typica multiphoton pathological diagnostic features of breast cancer.Multiphoton microscopy can identify cellular-level features, such as tumor cells, blood cells, adipose cells, myoepithelial cells, and lymphocytes on a small scale.These correspond to phenomena such as blood vessels invaded by tumor, microvessel proliferation 98 , tumor invasion of adipose tissue, microinvasive carcinoma 82 , and tumors surrounded by lymphocytes 24 on a larger scale.Additionally, it is possible to identify extracellular matrix-level features, such as collagen fibers and basement membrane on a small scale, which correspond to collagen fibrosis 99 and tumor breakthrough the basement membrane 23 on a larger scale    Fig. 4 Representative multiphoton pathological diagnosis algorithms.a Preprocessing is a necessary step to improve the accuracy of downstream diagnostic tasks, which is categorized into three types: virtual staining 17,140,141 , image restoration 73,134,135 , and super-resolution reconstruction 136,138 .b Diagnosis is divided into three categories: feature segmentation 28,29 , classification and identification [33][34][35]39 , and prognosis prediction 42 .Classification and identification are further subdivided into using deep learning models and traditional machine learning models.These categories encompass the input mode, model type, and output result.Adapted with permission from ref. 33,138,140   performance to human experts in certain tasks such as conventional H&E diagnostics.However, only a handful of these algorithms have been successfully integrated into standard clinical processes.As a result, the realization of effective artificial intelligence-based pathology diagnostics using multiphoton imagery remains fraught with challenges.As illustrated in Fig. 5, we will address these obstacles specifically associated with intelligent multiphoton pathology, examining the aspects of multiphoton imaging technologies, dataset acquisition, the development of deep learning algorithms, and their integration into clinical diagnostic procedures.

Multiphoton digital pathological diagnostic instrument
High-speed and high-throughput capability In comparison to clinical digital slide scanners, current multiphoton microscopes are still limited by imaging speed, cost, and image field of view.Particularly, there is an urgent need for a novel pathology imaging instrument with high-speed and high-throughput capabilities, similar to that of a digital slide scanner.The imaging speed of current multiphoton microscopes is primarily attributed to the scanning speed of two-axis mechanical scanning mirrors and the precision of motorized positioning stages.However, compared to digital slide scanner, the primary challenge lies in the sample preparation process for unstained slices used in multiphoton imaging.This process needs further optimization, such as adjusting slice thickness and adhering to sealing standards.Therefore, it is essential to standardize the quality of label-free slices based on objective working distance or excitation power of laser.This standardization is a prerequisite for improving the imaging throughput of multiphoton instruments.On the other hand, high-throughput simultaneously presents challenges in data transfer and storage.Currently, there are variations in data formats of commercial microscopes from different companies.Consequently, standardizing the multiphoton file formats for specific image compression protocols not only facilitates large-scale data storage but also promotes image sharing and consultation among pathologists.

Miniaturized and portable design
Research-grade multiphoton imaging instruments typically feature multifunctional characteristics, such as tunable femtosecond lasers, high-resolution spectrometers, and directly viewable eyepieces.However, some of features may be redundant for clinical applications.Therefore, multiphoton microscopes tailored for clinical pathology need to simplify the functionalities of researchgrade desktop multiphoton microscopes.This simplification not only enhances system usability but also effectively reduces the overall system size, weight, and manufacturing costs.It also makes multiphoton pathology microscopes more affordable and accessible to a broader range of medical centers and researchers, promoting their global adoption.Furthermore, miniaturized multiphoton pathology microscopes offer enhanced mobility and flexibility.In contrast to large-scale research-grade equipment, portable multiphoton microscopes do not require specific environments like cleanrooms for operation.This makes them more suitable for various clinical environments and applications, such as postoperative diagnosis in pathology departments, rapid intraoperative diagnosis in operating rooms, and bedside diagnosis in hospital wards.Notably, compared to conventional optical microscopes in pathology departments, the expense of miniaturized multiphoton microscopes remains considerable.This is primarily attributed to the costs associated with precision equipment, including femtosecond lasers, high numerical aperture objectives, and photomultiplier tubes.Therefore, although miniaturization facilitates the clinical integration of MPM, the substantial upfront investment and ongoing maintenance expenses frequently influence hospitals' procurement decisions.Such factors may impede the widespread adoption and collaborative utilization of MPM within medical institutions, ultimately diminishing its overall equipment utilization rates.

Multi-modality functionality
In clinical decision-making, the comprehensive utilization of multi-modal information is crucial for a more holistic understanding of diseases, encompassing clinical data and the combination of different imaging modalities such as radiology and pathology.For multiphoton microscopes, in addition to the four nonlinear optical effects (SHG, THG, 2PEF, 3PEF), SRS and coherent antistokes Raman scattering (CARS) also exhibits high specificity for different types of biomolecules.The integration of MPM and SRS/CARS in multimodal microscopy enables a more comprehensive characterization of the distribution of pathological features in tissues 143 .This not only aids in the discovery of novel pathological markers but also provides insights into disease mechanisms.On the other hand, H&E staining are one of the most used diagnostic tools in pathology.Encouragingly, H&Estained specimen can also be excited to produce multiphoton signals.Therefore, if H&E-stained imaging can be integrated with multiphoton imaging at the image or instrument level, it not only provides more comprehensive information during diagnosis but also enhances the reliability and accuracy of pathological diagnosis.Importantly, the integration of H&E staining establishes a more solid foundation for the widespread application of multiphoton pathology instruments in clinical settings.

Task-oriented high-quality open-source multiphoton datasets Focusing on specific clinical tasks
As multiphoton instruments are not yet widely employed in clinical pathology, the current scale of multiphoton image datasets is far smaller than that of digital pathology datasets.However, the effectiveness and utility of datasets are prerequisites for expanding dataset scale.To harness the unique advantages of multiphoton pathology diagnosis, it is imperative to establish taskoriented multiphoton pathology datasets, such as those for distinguishing brain tumors from pituitary tumors.Driven by specific clinical tasks, surgeons, pathologists, microscopists, and computer engineers need to collaboratively plan inclusion criteria, case numbers, image dimensions, annotation rules from the early stages of model development.This collaboration is essential to avoid biases that may impact model training.These taskoriented multiphoton datasets not only attract computer vision researchers to improve model metrics, but also draw more attention from clinical practitioners to the auxiliary diagnostic potential of MPM.

Image quality of dataset
Due to influences from factors such as photomultiplier gain, laser power, and sample preparation quality, even the same tissue slices may exhibit resolution and color discrepancies in images scanned by different multiphoton instruments.Such differences in image quality pose challenges to the transferability of the same model between two seemingly similar multiphoton datasets.Despite the development of some style normalization or style transfer models, these models often achieve optimal performance only on specific datasets.Therefore, AIassisted multiphoton pathology diagnosis should emphasize the rationalization of imaging parameters, standardization of imaging processes and specimen preparation.By exploring and establishing a consensus on the entire process from specimen to imaging, we may be able to control the quality of multiphoton image data from the source, thus addressing the generalization gap caused by inherent heterogeneity in histopathological data.

Open sourcing and sharing of dataset
Currently, acquiring multiphoton image still faces challenges, primarily due to the high academic value of multiphoton datasets and legal or ethical constraints involving human samples.It is worth noting that the rapid development of computer vision is closely related to the open-source and large-scale natural image datasets.To further propel the impact of multiphoton-assisted diagnosis, high-quality work should proactively release the datasets required for training models as much as possible, especially training data.This will prevent researchers from overestimating the performance of the models.Furthermore, to promote the sharing of large-scale datasets, we need to establish a network platform supporting online preview and download of multiphoton image data.This platform should include raw data, corresponding pathological images, dataset descriptions, and task instructions.On the other hand, to address challenges in sharing data when constructing multicenter datasets across different countries due to ethical and regulatory obstacles, federated learning and swarm learning can be attempted to jointly train the models.Federated learning allows multiple institutions to collaboratively improve a global model while preserving the confidentiality of their individual data sets.In parallel, swarm learning enhances prediction accuracy and robustness by integrating diverse models.This approach effectively mitigates overfitting and enhances the model's generalization capabilities.

Custom-developed multiphoton deep-learned diagnostic tool
Transitioning from supervised to unsupervised training paradigm Supervised, unsupervised, and semi-supervised learning are the three main training paradigms in deep learning.Supervised learning relies on experts annotating multiphoton images, but obtaining paired ground truth can be challenging.Computational constraints often lead to training gigapixel or terapixel-level images with annotated patches, which is time-consuming and expensive.Moreover, models trained on a single dataset usually lack strong generalization.Self-supervised learning addresses this by designing supervision tasks that transform unsupervised learning into a supervised problem without requiring manual annotations, while semi-supervised learning leverages a small amount of labeled data alongside unlabeled data to reduce dependency on extensive labeling.In segmentation tasks, a self-supervised domain adaptation framework, based on target-specific fine-tuning, adapts the original model to different target-specific pathological tissues for cell segmentation.This domain adaptation occurs across various tissues and multiple medical centers without accessing the source dataset, enhancing the model's performance even with minimal labeled data 144 .Additionally, a semi-supervised semantic segmentation network, SCANet, based on a three-branch architecture, alternately trains a multi-scale recurrent neural network branch, a consistency decoder branch, and an adversarial learning branch.This achieves excellent segmentation performance with a small amount of labeled data and extensive unlabeled data 145 .
Weakly supervised learning harnesses imprecise or incomplete weak label information to train models, mapping input data to stronger labels, thereby reducing reliance on precise annotations.In classification tasks, a weakly supervised learning framework using Information Bottleneck theory fine-tunes the backbone to create taskspecific representations from WSI-level weak labels, addressing the limited annotation issue in pathological image classification 146 .Similarly, another weakly supervised learning framework based on RankMix data augmentation, adapts sample quantities in the training set according to task contributions and mix images of different sizes, mitigating issues of data scarcity and class imbalance 147 .Ultimately, self-supervised or weakly supervised learning holds promise in addressing challenges such as inadequate generalization, data scarcity, and insufficient labeled data in multiphoton pathology models.

Model architecture for general intelligence
Model performance metrics reflect their ability to perform tasks on specific datasets.From a diagnostic perspective, pathologists are equally concerned about the intelligence of the model's adaptability and handling of boundary conditions.Firstly, introducing advanced model architecture is crucial for the future of multiphoton intelligent diagnostics.Unlike CNN models trained on small patient cohorts, combining a pre-trained encoder with a transformer network for patch aggregation has been validated for end-to-end biomarker prediction on a large multicenter cohort of over 13,000 colorectal cancer patients 148 .On the other hand, the process of multiphoton imaging is interpretable, where the pixel intensity in the image represents the spectral characteristics of endogenous fluorescence signal sources.Thus, incorporating the physical principles of MPM into the model ensures more effective capture of endogenous information, potentially revolutionizing the interpretation of multiphoton data and enhancing both generalizability and efficiency.
Secondly, a single modality often fails to fully reveal the complex mechanisms and diversity of diseases, medical centers have established multidisciplinary teams for the clinical treatment of major illnesses.Moreover, molecular pathology laboratories equipped with technologies such as genetic testing, protein analysis, and fluorescence imaging are increasingly demonstrating their capacity for precise diagnosis.Therefore, AI models that integrate multimodal data can provide comprehensive and scientifically sound diagnostic decisions.The histological and genomic features are extracted using a multiple instance learning network and a self-normalizing network, followed by feature fusion through Kronecker product integration to achieve cancer prognosis prediction 149 .The iStar model, which is based on hierarchical image feature extraction, combines spatial transcriptomics data with highresolution histological images to predict superresolution spatial gene expression 150 .Pan-cancer computational histopathology represents image tile as 1536dimensional vectors and uses high-dimensional regression methods to integrate histological, genomic, and transcriptomic features, accurately discriminates 28 cancer and 14 normal tissue types 151 .As a result, incorporating multiphoton image features into the multimodal AI models has the potential to offer unique new perspectives on the interactions between cells and the extracellular matrix within the tumor microenvironment.
Finally, foundational models like ChatGPT in natural language processing demonstrate capabilities for general intelligence, facilitating the development of multiphoton diagnostic models with multitasking abilities.Future advancements will enable tasks such as transforming between multiphoton and H&E images, interpreting multiphoton images alongside pathological reports, and engaging in iterative question-and-answer sessions involving pathological findings and doctor-patient interactions 152 .A more challenging prospect is transforming multiphoton microscopes into intelligent entities through specialized models, allowing interaction with highthroughput images in clinical pathology diagnostics.This embodied intelligent learning paradigm will ultimately lead to new emergences in MPM diagnostic capabilities, providing opportunities to construct a general intelligent model adaptive to diseases.

Interpretability, repeatability and reliability
Although some multiphoton diagnostic models perform exceptionally well on datasets, even matching or surpassing human diagnostic tasks, the primary hurdle in clinical application is the "black-box" nature of deep learning, i.e., lack of interpretability.Pathologists express concern about writing diagnostic reports when they lack an understanding of how the model reaches its conclusions.Despite interpretability of neural networks has been a long-standing challenge, the methods like feature visualization could provide an approximate explanation of the model's working process.These visualization results enhance pathologists' trust in model-assisted decisionmaking.On the other hand, the reliability of auxiliary diagnostics is also reflected in the model's repeatability.In the field of multiphoton medicine, while extensive work has been done on medical statistical analysis or model ablation experiments, open-source code contributions are limited.For the open-source work, the training weights of the model are particularly crucial for reproducing results.Therefore, if we verify the repeatability of the model through sufficient code access privileges and data resources, thereby providing confidence intervals, capability boundaries, and computational consumption.This will increase the reliability of the model for clinical deployment.However, achieving breakthroughs in AI interpretability poses significant challenges in the short term.If guided by outcome-driven assessments of model feasibility, clinical validation of deep learning methods emerges as a crucial pathway to enhancing AI reliability, particularly in healthcare settings.For instance, within large-scale multicenter trials employing AI-empowered MPM, despite lingering uncertainties regarding the interpretability of the models, the accuracy metrics of diagnostic tasks serve as robust indicators of their reliability and stability.This, in turn, will also enhance patient acceptance of this novel technology.

The clinical workflow of integrated multiphoton pathology Multiphoton pathological diagnostic criteria
Pathologists, drawing upon years of accumulated knowledge and experience, have established standard criteria for conventional pathological diagnosis.Even though MPM has demonstrated a series of advancements in pathological applications, firstly, it is essential to establish atlases tailored to multiphoton diagnosis.These atlases should elucidate the diverse applications of multiphoton images across various pathological scenarios.They ought to encompass multiphoton images alongside corresponding images of fresh tissue, frozen sections, paraffin-embedded sections, smears, and organoids for comparative reference.Such comprehensive coverage will assist pathologists in gaining deeper insights into MPM indications and serve as an introductory guide for computer vision researchers exploring multiphoton imaging.Secondly, pathologists typically have minimal or no training in the use of multiphoton-assisted diagnostic technologies.To facilitate a rapid understanding of multiphoton images by pathologists, a virtual staining model can be employed.Multiphoton images can be transformed into virtual H&E images, special stains, and even holds the potential for conversion into immunohistochemistry or immunofluorescence images 153 .This capability allows pathologists to engage in paired comparative learning, assisting them in gradually incorporating multiphoton features into their diagnostic workflow.With the growing trust among pathologists in multiphoton diagnostics, multi-center clinicians can continuously validate and explore new multiphoton features in clinical practice.This iterative process allows for the enhancement of multiphoton diagnostic capabilities across various medical settings.Finally, combined with efficient AI analysis, this approach can further aid in formulating comprehensive pathology workflows and improving diagnostic precision.For instance, Pohlkamp et al. investigated the use of machine learning to support microscopic differential counts of peripheral blood smears within a highthroughput hematology laboratory setting 154 .Nasrallah et al. utilized machine learning for cryosection pathology to predict the 2021 WHO classification of glioma 155 .As multiphoton diagnostic methods achieve consensus, it is anticipated that clinicians and imaging experts will collaboratively integrate multiphoton diagnostic features into clinical diagnostic guidelines or novel histological grading systems for specific diseases.

Multiphoton pathological diagnosis platform
The prospect of multiphoton AI-assisted diagnostic algorithms is exciting for pathologists.However, pathologists typically a background in computer science, and reproducing algorithms or configuring environments can be labor-intensive for them.In the pathology diagnostic workflow, pathologists prefer "plug-and-play" intelligent diagnostic software for decision support.Therefore, there is an urgent need to integrate mature multiphoton diagnostic algorithms into pathology diagnostic systems, such as picture archiving and communication system, in the form of software packages.Pathologists can seamlessly import MPM-based diagnostic results into conventional diagnostic reports, facilitating easier integration into existing diagnostic workflows.Additionally, using cloudbased interactions, pathologists can collaboratively assess this novel multiphoton pathology report with colleagues, with final decisions made by senior pathologists.For less mature models, there is a need for a research-level specialized diagnostic platform for multiphoton images, similar to DeepImageJ 156 .This platform should deploy and fine-tune pre-trained deep learning models, creating a library of multiphoton diagnostic algorithms.Based on various postoperative or intraoperative diagnostic requirements, algorithms from the platform can be selectively deployed to edge or cloud servers.Besides, a feedback mechanism should be incorporated into the software process to iteratively optimize the diagnostic performance of algorithms in clinical trials.

Ethical security and AI risks
While interdisciplinary personnel have considered ethical security concerns in constructing multiphoton image datasets, multiphoton images involve patient privacy information.In the data management and analysis processes of multiphoton diagnostic software, apart from pathologists, it may also involve bioinformatics, statistics, and computer vision researchers.This may inadvertently lead to risks related to personal privacy or the illegal utilization of data.To address these issues, it is essential to establish privacy protection mechanisms and data protection regulations concerning multiphoton-related data.Beyond ethical security, AI risks also demand attention.Data poisoning and adversarial sample attacks are common methods that threaten the security of the model.Data poisoning involves injecting samples or altering data in the training set to deceive the model, leading to incorrect predictions in future tasks.Adversarial attacks, on the other hand, subtly but purposefully modify input data to cause the model to produce incorrect results.The augmentation of multiphoton data significantly enhances the model's inference capabilities.However, this increased capability may introduce false features that clinicians find challenging to identify.For instance, the results of virtual staining, without a ground truth for comparison, already poses challenges for pathologists in distinguishing between authentic and synthetic information.This uncertainty can introduce decision biases for pathologists, with the impact on patient prognosis difficult to estimate.To mitigate these AI risks and reduce uncertainty in diagnosis, perhaps there is no need to blindly pursue the innovation and performance of the model; instead, emphasis should be placed on the practicality and stability of the model.Additionally, multiphoton diagnostic software requires rigorous clinical validation and regulatory approval.Through randomized clinical trials, it can determine the role of multiphoton diagnostic algorithms in the entire diagnostic workflow.This ensures the provision of more reliable, controllable, and secure diagnostic results.

Conclusion
Despite the hurdles in progressing multiphoton microscopy (MPM) from traditional pathological uses to intelligent diagnostics, the movement toward smart multiphoton pathology is actively underway.As multiphoton pathology tools evolve and the collection of relevant datasets grows, we anticipate a marked enhancement in both the breadth and depth of artificial intelligence applications within this field.Pathologists are beginning to grasp the enhanced capabilities offered by multiphoton technology.However, it is important to stress that the successful implementation of such sophisticated technology hinges on synchronized collaborative efforts from diverse, interdisciplinary teams across multiple centers.This cooperation is vital for turning scientific discoveries into actionable diagnostic criteria, for refining early-stage prototypes into approved medical devices, and for evolving open-source algorithms into accessible, usercentered software interfaces.With these concerted efforts, MPM is poised to become a cornerstone in the future landscape of diagnostic pathology.

Fig. 5
Fig. 5 Structural diagram of AI-empowered multiphoton pathology.The multiphoton instrument and multiphoton dataset serve as the cornerstone of multiphoton pathological diagnosis, laying the foundation for the development of intelligent diagnostic tools.These tools are developed based on advanced model architectures and training paradigms, possessing functions such as image preprocessing, disease diagnosis, and prognosis prediction, ultimately integrated into an intelligent diagnostic platform.By integrating the multiphoton atlas and diagnostic criteria into clinical workflow, AI-empowered multiphoton pathology promotes the establishment of a paradigm for multiphoton pathological diagnosis

Table
Comparison of label-free biomedical microscopy

Table 2 (
a) Multiphoton imaging parameters and pathological characteristics of tumor diseases.(b) Multiphoton imaging parameters and pathological characteristics of non-tumor diseases

Table 3 (
a) Image preprocessing algorithms for multiphoton microscopy.(b) Diagnosis algorithms for multiphoton microscopy